Nucleic acid detection combining amplification with fragmentation

ABSTRACT

Provided herein are methods and compositions for detection of a nucleic acid target in a sample. The methods and compositions use primer directed amplification in conjunction with nucleic acid fragmentation. The methods have high sensitivity even in the presence of a large amount of non-target nucleic acid. Also provided are oligonucleotides and kits useful in the method. Exemplary nucleic acid targets are those with mutant gene sequence such as mutant sequence of the EGFR, APC, TMPRSS2, ERG and ETV1 genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.15/712,448, filed Sep. 22, 2017, which is a Divisional of U.S.application Ser. No. 14/679,403, filed Apr. 6, 2015, which is aContinuation of U.S. application Ser. No. 12/035,356, filed Feb. 21,2008, which claims priority to U.S. Provisional Patent Application No.60/926,611, Titled: Nucleic Acid Detection Combining Amplification WithFragmentation, filed Apr. 27, 2007 and U.S. Provisional PatentApplication No. 61/007,928, Titled: Nucleic Acid Detection CombiningAmplification With Fragmentation, filed Jun. 8, 2007, which areincorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 18, 2018, isnamed sequence.txt and is 17 KB.

FIELD OF THE INVENTION

Provided herein are methods and compositions for detecting targetnucleic acid such as mutant nucleic acid. The methods and compositionscombine amplification with nucleic acid fragmentation, are useful fordetecting very low amounts of target nucleic acids, even in the presenceof large amounts of non-target nucleic acids.

BACKGROUND OF THE INVENTION

Although nucleic acid assays are known to offer a high degree ofspecificity, there are limits in the sensitivity of such assays,particularly when the target nucleic acid to be detected is present inrelatively low quantities compared to non-target nucleic acid. In thecase of cancer, the ability to detect the presence of a small amount ofa cancer specific mutant nucleic acid allows for early cancer diagnosisand offers the possibility of more effective therapeutic intervention.However, detection can be challenging if the sample of nucleic acidbeing tested has very little of the mutant nucleic acid and if there isan excess of normal nucleic acid in the sample. Although a tumor biopsymay contain significant mutant nucleic acids, a plasma sample from acancer patient may contain one or only a few copies of a mutant nucleicacid of interest. Amplification methods such as PCR may detect a fewcopies of a mutant nucleic acid, however, the abundance of normalnucleic acid in samples such as plasma can interfere.

Focus has been placed on identifying tumor-derived mutations incirculating DNA found in plasma or serum of solid tumor patients as anoninvasive and early diagnostic tool. Confirmed reports of the presenceof solid tumor-derived mutations found in circulating DNA include, butare not limited to, patients with colorectal tumors, pancreatic cancer,breast cancer, head and neck squamous cell carcinoma, and lung cancer(Hibi et al. 1998, Chen et al. 1999, Diehl et al. 2005, Coulet et al.2000, Hagiwara 2006, Kimura et al. 2006).

Reports have also demonstrated that cancer patients show elevated levelsof circulating DNA and have proposed use of DNA quantification asprognostic and diagnostic factors (Gautschi et al. 2004, Goebel et al.2005, Sozzi et al. 2001, Herrera et al. 2005, Pathak et al. 2006). Thishas led to efforts to describe the origin of such elevated levels ofDNA. While still under investigation, the sharp increase in circulatingDNA is not likely attributed to DNA released from tumor cells. In fact,analysis of mutations present in the plasma of patients with colorectaltumors revealed that the levels of mutations found in circulating DNAdid not increase proportionally with the overall elevated levels ofcirculating DNA (Diehl et al. 2005). Thus while some cancer patientsshow elevated levels of plasma DNA, detection of tumor-derived mutationswill require the ability to detect very few mutations in the presence oflarger amounts of wild type DNA.

A number of strategies have been described for detecting low copy numbernucleic acid targets. Methods including allele-specific PCR of p53 andABL kinase domain mutations have demonstrated sensitivities ranging from0.1-0.01% and in one mutation, 0.001% (Righetti et al. 1999, Coulet etal. 2000, and Kang et al 2006). Ohnishi, H., et al. reported a method ofamplification using a mutation specific primer that spans a deletionsite and does not anneal to the wild-type sequence. Ohnishi, H., et al.,15(2) Diagnostic Molecular Pathology 101-108 (2006). Mutation specificprimers of the Scorpion type also have been reported. Kimura, H. et al.,12(13) Clinical Cancer Research 3915-3921 (2006); Newton, C. R., et al.,17(7) Nucleic Acids Research 2503-2516 (1989); and Whitcombe, D. et al.,17 Nature Biotechnology 804-807 (1999) (describing Scorpion ARMS primersand strategies for primer design). Methods that enrich mutant nucleicacid by digesting wild-type DNA with restriction enzymes prior toamplification have been reported. Asano, H., et al., 12(1) ClinicalCancer Research 43-48 (2006); Gocke, C., et al., U.S. Pat. No.6,630,301. The Asano et al., method uses multiple PCR reactions. A firstPCR reaction is used to remove an upstream restriction enzymerecognition site. Following the first PCR, a restriction digestion isperformed. After digestion, a second PCR reaction is used to amplify thetarget sequence.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions for detecting targetnucleic acids at very low levels and in the presence of large amounts ofnon-target nucleic acids. Generally, a target and non-target nucleicacid are distinguished by the presence or absence of a fragmentationsite, such as a restriction enzyme recognition site. By differentiatingthe target and non-target by a fragmentation site, the methods andcompositions used herein can be used with various nucleic acid detectionmethods known in the art, such as PCR.

As used herein, the term “target” nucleic acid refers to a nucleic acidwhich contains an allele or a mutant nucleic acid sequence. A mutantnucleic acid sequence may be any mutant sequence including but notlimited to substitution, insertion, deletion, and translocation.

As used herein, the term “non-target” or “other” nucleic acid used inreference to a target nucleic acid means a nucleic acid that does notcontain the target sequence. For example, a non-target nucleic acid of atarget nucleic acid encoding an allelic sequence encompasses nucleicacid that contains an alternative allele. The non-target of a nucleicacid containing a mutant sequence is a nucleic acid that contains normalor wild-type nucleic acid sequence with respect to the mutant sequence.

As used herein, the term “locked nucleic acid” or “LNA” refers tobicyclic nucleic acid analogs contain one or more 2′-O, 4′-C methylenelinkage(s), which effectively locks the furanose ring in a C3′-endoconformation. This methylene linkage restricts the flexibility of theribofuranose ring and locks the structure into a rigid bicyclicformation. Because of its structural conformation, locked nucleic acidsdemonstrate a much greater affinity and specificity to theircomplementary nucleic acids than do natural DNA counterparts andincreases the thermal and chemical stability of a primer/target nucleicacid duplex. LNAs will hybridize to complementary nucleic acids evenunder adverse conditions, such as under low salt concentrations and inthe presence of chaotropic agents. According to one aspect of theinvention, locked nucleic acids increase the melting point of theprimer/target nucleic acid duplex by about 3 to about 8° Celsius perlocked nucleic acid base incorporated in the primer. The basicstructural and functional characteristics of LNAs and related analoguesare disclosed in various publications and patents, including WO99/14226, WO 00/56748, WO 00/66604, WO 98/39352, U.S. Pat. Nos.6,043,060, and 6,268,490; see also, Braasch et al., “Locked nucleic acid(LNA): fine-tuning the recognition of DNA and RNA,” Chem. Biol. 8(1):1

Locked nucleic acid bases may be interspersed throughout a strand of aprimer, placed consecutively or placed singularly in predeterminedlocations. In one embodiment, the mutation specific primer comprises alocked nucleic acid base at its 3′ terminus. In another embodiment, themutation specific primer comprises a locked nucleic acid base at its N−1(i.e., penultimate) base. The mutant base may be a locked nucleic acid.

In one aspect, provided herein is a method for detecting the presence orabsence of a target nucleic acid by testing a sample that potentiallycontains the target nucleic acid in the presence of non-target nucleicacid, the method includes: a) fragmenting the sample nucleic acid underconditions such that a subsequent amplification directed to the targetnucleic acid results in an increased detection of the target nucleicacid over the non-target nucleic acid as compared to amplificationwithout fragmentation; b) amplifying the target nucleic acid with a pairof primers, where a first primer is specific for the target nucleicacid; and c) detecting the presence or absence of an amplificationproduct, which indicates the presence or absence of the target nucleicacid in the sample.

In another aspect, provided herein is a method for diagnosing a canceror detecting the presence of a tumor cell by determining if anindividual has a mutant sequence associated with the cancer or tumorcell type, the method includes: a) obtaining a sample including nucleicacid from the individual; b) fragmenting the sample nucleic acid underconditions such that a subsequent amplification directed to the targetnucleic acid results in an increased detection of the target nucleicacid over the non-target nucleic acid as compared to amplificationwithout fragmentation; c) amplifying the target nucleic acid with a pairof primers, where a first primer is specific for the target nucleicacid; and d) detecting the presence or absence of an amplificationproduct containing the mutant sequence, where diagnosis of cancer isdetermined by the presence absence or amount of amplification productcontaining the mutant sequence.

In yet another aspect, provided herein is a method for determiningprognosis with cancer by determining if an individual has a mutantsequence associated with the cancer, the method includes: a) obtaining asample containing nucleic acid from the individual; b) fragmenting themutant nucleic acid under conditions such that a subsequentamplification directed to the mutant nucleic acid results in anincreased detection of the mutant nucleic acid over the non-mutantnucleic acid as compared to amplification without fragmentation; c)amplifying the mutant nucleic acid with a pair of primers, where a firstprimer is specific for the mutant nucleic acid; and d) detecting thepresence, absence and/or amount of an amplification product containingthe mutant sequence, where the likelihood of an outcome in theindividual is associated with the presence and or amount of mutantnucleic acid sequence.

In still yet another aspect, provided herein is a method for determiningdrug sensitivity of an individual diagnosed with cancer, the methodincludes: a) obtaining a sample comprising nucleic acid from theindividual; b) fragmenting the mutant nucleic acid under conditions suchthat a subsequent amplification directed to the mutant nucleic acidresults in an increased detection of the mutant nucleic acid over thenon-mutant nucleic acid as compared to amplification withoutfragmentation; c) amplifying the mutant nucleic acid with a pair ofprimers, where a first primer is specific for the mutant nucleic acid;d) detecting the presence, absence and/or amount of an amplificationproduct containing the mutant sequence; and e) relating the presence,absence and/or amount of an amplification product containing the mutantsequence to cancer drug sensitivity. Some examples of mutations thataffect drug sensitivity which may be targeted by the assay methodsdescribed herein are described in Lynch, et al., 350(21) NEJM 2129-2139(2004); Bell, et al., US Patent App. No. 20060147959 (2005) (determiningtyrosine kinase inhibitor, i.e., gefitinib and erlotinib sensitivity bydetecting EGFR mutations); and Sawyers, et al., U.S. Patent Appl. No.2006/0269956 (describing mutations that affect drug resistance toBCR-ABL kinase activity inhibitors typically used to treat CML due tothe T315I mutation in the Abl gene).

In certain embodiments of the aspects provided herein, the mutatednucleic acid sequence is due to a deletion, insertion, substitutionand/or translocation or combinations thereof. In preferred embodiments,fragmentation of nucleic acid sequence in which cleavage of wild-typesequence is with a restriction enzyme. Such pre-amplification digestiontreatment allows for fragmentation to destroy or substantially decreasethe number of wild-type sequences that might be amplified. In yet morepreferred embodiments, the fragmentation using a restriction enzyme iscombined with the use of a mutation specific primer (or mutated sequenceprimer).

In preferred embodiments, a mutated sequence destroys or disrupts arestriction enzyme recognition site present in the correspondingwild-type sequence and that a mutation specific primer can be designedto bind to the mutated version of the sequence and not its wild-typecounterpart. For example a mutation specific primer can overlap a borderregion, which is a region that contains portions of both a wild-typesequence adjacent to a portion of the mutated sequence. In furtherexamples, if a mutation is the result of a deletion, such as the 15 bpdeletion in exon 19 of the Epidermal Growth Factor Receptor (EGFR) gene(E746_A750del), a mutation specific primer could be designed, asillustrated in FIG. 1, so as to span a new site in the DNA which arisesfrom the deletion. Other methods of detecting EGFR nucleic acid aredescribed in U.S. Pat. No. 6,759,217 (which describes detecting EGFRnucleic acid in plasma or serum), U.S. Pat. Nos. 6,127,126 and 5,981,725(both disclose detecting nucleic acid encoding an EGFR mutant proteintype II for a mutation in which a portion of the extracellular domain ofEGFR is deleted). If a mutation is due to an insertion, a mutationspecific primer could be designed to span either or both junctions wherethe inserted sequence is adjacent to wild-type sequence. If a mutationis due to one or more substitutions, then a mutation specific primercould be designed to span any or all of the substitutions. If a mutationis due to a translocation, then a mutation specific primer could bedesigned to span one or both junctions of the translocated sequence, inany region where the sequence is altered by the translocation. Theseexamples are merely exemplary and provide guidance to one of skill inthe art to design various permutations of primers that would anneal to amutated sequence and not a wild-type sequence which are appropriate forthe methods and compositions provided herein.

In one approach, a sample is assayed for the presence or absence of amutated sequence by amplification and detection of the resultingamplification products. In a preferred embodiment, amplification oftarget nucleic acids is accomplished by polymerase chain reaction (PCR).

Single or multiple mutant sequences can be assayed. Amplification ofmultiple mutant sequences can be performed simultaneously in a singlereaction vessel, e.g., multiplex PCR. In this case, probes may bedistinguishably labeled and/or amplicons may be distinguishable by sizedifferentiation. Alternatively, the assay could be performed in parallelin separate reaction vessels. In such later case, the probes could havethe same label.

In certain embodiments of the aspects provided herein, the methodsfurther comprise a nucleic acid extraction step. Various extractionnucleic acid methods are known in the art which can be employed with themethods and compositions provided herein such as lysis methods (such asalkaline lysis), phenol:chloroform and isopropanol precipitation.Nucleic acid extraction kits can also be used. Preferably, theextraction method is Agencourt Genfind™, Roche Cobas® orphenol:chloroform extraction using Eppendorf Phase Lock Gels®. Morepreferably the nucleic acid is extracted using Agencourt Genfind™

Also provided are exemplary oligonucleotides useful in the methods andkits described herein.

The following mutated sequences can be detected with the methods andcompositions provided herein. The methods outlined for detection ofspecific deletion mutations, insertion mutations, point mutations andfusion transcripts can be applied to any biomarker which may be usedwith fragmentation, particularly when a restriction digestionrecognition site is disrupted. Embodiments of specific primer designsare described below but the sequence will vary to fit the mutation to bedetected. Restriction enzyme digestion sites will also depend on thesequence of the wild-type sequence as compared to the mutated nucleicacid or fusion transcript. The frequency of various restriction sitesfound in DNA virtually ensures that a site unique to the wild-type DNAof interest can be found for any mutation detection assay, thus thismethodology is applicable to a wide array of cancer biomarkers.

In one approach, a mutation specific primer is designed for detecting adeletion mutation. Mutation specific primer can be designed to span thedeleted region such that the primer contains wild-type sequence thatlies 5′ and 3′ of the deleted region or the complement thereof. Thus,the mutation specific primer cannot bind to the wild-type sequence andcannot produce an amplicon.

In one approach, a mutation specific primer is designed for detecting aninsertion mutation. A mutation specific primer can be designed to spanall or a portion of the inserted region such that the primer includesall or a part of the inserted region. A primer could be designed to spanthe either or both junctions of the inserted sequence, for example, theprimer sequence would include a portion of wild-type sequence that isadjacent to the inserted sequence or the complement thereof. Thus, themutation specific primer is not complementary to the wild-type sequenceand cannot produce an amplicon.

In one approach, a mutation specific primer is designed for detectingone or more substitution mutations. A mutation specific primer can bedesigned to include one or more substitutions or the complement thereof.For example, the 3′ nucleotide of the primer can be designed such thatit contains the mutated base pair and does not bind hybridize, or basepair, in the wild-type gene and thus cannot elongate.

In one approach, a mutation specific primer is designed for detectingone or more translocation mutations. A mutation specific primer can bedesigned to span the junction of the translocation or the complementthereof. A primer pair could be designed to so that one primer isupstream of the translocation junction and the second is downstream ofthe junction. Thus, when the primer pair is used on wild-type sequence,no amplification products will be produced because the locations of theprimers relative to each other are cannot be amplified. However, whenthe translocation is present, the primers are in close enough proximityof each other such that an amplification product can be produced. Forexample, the primer can be designed to include a portion of the firstgene and a portion of the second gene, where the genes are located ondifferent chromosomes in wild-type form but are adjacent to one anotherin the mutated form.

In certain embodiments, at least one primer of each primer pair in theamplification reaction is labeled with a detectable moiety. Thus,following amplification, the various target segments can be identifiedby size and color. The detectable moiety is preferably a fluorescentdye. In some embodiments, different pairs of primers in a multiplex PCRmay be labeled with different distinguishable detectable moieties. Thus,for example, HEX and FAM fluorescent dyes may be present on differentprimers in multiplex PCR and associated with the resulting amplicons. Inother embodiments, the forward primer is be labeled with one detectablemoiety, while the reverse primer is labeled with a different detectablemoiety, e.g. FAM dye for a forward primer and HEX dye for a reverseprimer. Use of different detectable moieties is useful fordiscriminating between amplified products which are of the same lengthor are very similar in length. Thus, in certain embodiments, at leasttwo different fluorescent dyes are used to label different primers usedin a single amplification. In still another embodiment, control primerscan be labeled with one moiety, while the patient (or test sample)primers can be labeled with a different moiety, to allow for mixing ofboth samples (post PCR) and the simultaneous detection and comparison ofsignals of normal and test sample. In a modification of this embodiment,the primers used for control samples and patient samples can be switchedto allow for further confirmation of results.

Analysis of amplified products from amplification reactions, such asmultiplex PCR, can be performed using an automated DNA analyzer such asan automated DNA sequencer (e.g., ABI PRISM 3100 Genetic Analyzer) whichcan evaluate the amplified products based on size (determined byelectrophoretic mobility) and/or respective fluorescent label.

The methods and compositions provided herein provide increasedsensitivity for detection of a mutated nucleic acid. Preferably themethods can detect mutated nucleic acid that is present in 10% or less,1% or less, 0.1% or less, 0.01% or less, 0.001% or less, 0.0005% orless, 0.0003% or less, or 0.0002% or less than the total nucleic acid ofa sample.

Various other cancer biomarkers suitable for detection using the methodsand compositions provided herein include, but are not limited to, breastcancer markers, such as, GSTP1, RASSF1A (both described in Papadopoulou,E. et al., 1075 Ann. N.Y. Acad. Sci. 235-243 (2006); and RASSF1A(Papadopoulou, E., et al., and Coyle, et al., 16(2) Cancer Epidemiol.Biomarkers Prev. 192-196 (2007)), ATM (Papadopoulou, E., et al.), APC(Coyle, et al.), RARbeta2 (Hogue, M O, et al., 24(26) J. Clin. Oncol.4262-4269, Epub 2006 Aug. 14 (2006)), and TP53 (Silva, J. M., et al.,8(12) Clin. Cancer Res. 3761-3766 (2002)); ovarian cancer markers, suchas p53 (Swisher, E. M., et al., 193(3) American Journal of Obstetricsand Gynecology 662-667 (2005)); hepatocellular carcinoma markers, suchas, p53 mutations (Huang, X. H., et al., 9(4) World J Gastroenterol.692-695 (2003)), and p 16 (Le Roux, E., 53(3) Rev. Epidemiol. SantePublique. 257-266 (2005)); and pancreatic cancer markers, such as K-ras(Castells, A., et al., 17(2) J. Clin. Oncol. 578-584 (1999)).

Oligonucleotides or combinations of oligonucleotides that are useful asprimers or probes in the methods are also provided. Theseoligonucleotides are provided as substantially purified material.

Kits comprising oligonucleotides which may be primers for performingamplifications as described herein also are provided. Kits may furtherinclude oligonucleotides that may be used as probes to detect amplifiednucleic acid. Kits may also include restriction enzymes for digestingnon-target nucleic acid to increase detection of target nucleic acid bythe oligonucleotide primers.

As used herein, the term “junction” refers to the position where targetand non-target sequences are adjacent to one another due to a sequencechange. For example, in the event of a translocation between sequence 1,ATGC and sequence 2, CGTA, the resulting mutated sequence or fusionsequence would be ATGCCGTA. The junction in this translocation examplewould be “CC.” For example, in the even of an insertion of sequence 3,CCCC into sequence 4, ATGC, the resulting mutated sequence would beATCCCCGC. The junctions in this event would be “TC” and “CG.”

“Fragmentation” as used herein refers a process in which longer lengthsof nucleic acid are broken up into shorter lengths of nucleic acid.Nucleic acids may be broken up or fragmented by chemical or biochemicalmeans, preferably nucleic acids are fragmented in a manner that isreproducible, preferably nucleic acids are fragmented by one or morerestriction endonucleases. The length of a fragment containing thenucleic acid segment of interest can depend on the length of the nucleicacid segment of interest as well as the restriction enzyme chosen tofragment the DNA.

A “restriction endonuclease” or “restriction enzyme” as used hereinrefers to an enzyme that cuts double-stranded DNA at a specific sequence(i.e., the recognition sequence or site). The frequency with which agiven restriction endonuclease cuts DNA depends on the length of therecognition site of the enzyme. For example, some enzymes recognizesites that are four nucleotides long (referred to as “four cutters”). Ingeneral one can estimate how frequently an enzyme should cut a piece ofDNA based the length of the recognition site and the assumption that theprobability of any one nucleotide occurring at a given location is ¼. Inthe case of a “four cutter” a specific sequence of four nucleotides mustbe present. Assuming that each nucleotide has an equal chance (i.e., ¼)of occurring at any particular site within the four nucleotide sequence,then a four-cutter should on average cut once every 256 base pairs(i.e., ¼×¼×¼×¼= 1/256). A similar calculation can be applied to anyrestriction enzyme as long as the length of its recognition site isknown, making it possible to predict the size and number of a DNAfragments that would be obtained by cutting a DNA molecule of knownsize. This allows one of skill in the art to produce DNA fragments ofknown size. Restriction endonucleases are obtained from bacteria or areproduced through recombinant technology and are readily availablethrough numerous commercial sources.

As used herein, the term “increased detection” refers to the ability todetect lower amounts of target nucleic acid in the presence ofnon-target nucleic acid. For example, as non-target nucleic acidincreases, fragmentation of the non-target nucleic acid increases theability to detect a smaller fraction of target nucleic acid in totalnucleic acid.

As used herein, the term “sample” or “test sample” refers to any liquidor solid (or both) material can be used to test for the presence ofnucleic acids. In preferred embodiments, a test sample is obtained froma biological source (i.e., a “biological sample”), such as cells inculture or tissue cells from an animal, preferably, a human. Preferredsample sources include, but are not limited to, sputum (processed orunprocessed), bronchial alveolar lavage (BAL), bronchial wash (BW),blood, bone marrow, bodily fluids, cerebrospinal fluid (CSF), urine,plasma, serum or tissue (e.g., biopsy material). A body fluid samplerefers to fluid containing samples from an individual including sputum(processed or unprocessed), bronchial alveolar lavage (BAL), bronchialwash (BW), blood, plasma, serum, and cerebrospinal fluid (CSF). The term“patient sample” as used herein refers to a sample obtained from a humanseeking diagnosis and/or treatment of a disease.

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. Oligonucleotides are generally between about 10, 11, 12, 13, 14or 15 to about 150 nucleotides (nt) in length, more preferably about 10,11, 12, 13, 14 or 15 to about 150 nt, more preferably about 10, 11, 12,13, 14, or 15 to about 70 nt, and most preferably between about 20 toabout 26 nt in length. The single letter code for nucleotides is asdescribed in the U.S. Patent Office Manual of Patent ExaminingProcedure, section 2422, table 1. In this regard, the nucleotidedesignation “R” means guanine or adenine, “Y” means thymine (uracil ifRNA) or cytosine; and “M” means adenine or cytosine. An oligonucleotidemay be used as a primer or as a probe.

As used herein, the term “detecting” used in context of detecting asignal from a detectable label to indicate the presence of a targetnucleic acid in the sample does not require the method to provide 100%sensitivity and/or 100% specificity. As is well known, “sensitivity” isthe probability that a test is positive, given that the person has atarget nucleic acid sequence, while “specificity” is the probabilitythat a test is negative, given that the person does not have the targetnucleic acid sequence. A sensitivity of at least 50% is preferred,although sensitivities of at least 60%, at least 70%, at least 80%, atleast 90% and at least 99% are clearly more preferred. A specificity ofat least 50% is preferred, although sensitivities of at least 60%, atleast 70%, at least 80%, at least 90% and at least 99% are clearly morepreferred. Detecting also encompasses assays with false positives andfalse negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% oreven higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or evenhigher.

As used herein, the term “substantially purified” in reference tooligonucleotides does not require absolute purity. Instead, itrepresents an indication that the specified oligonucleotide isrelatively more pure than it is in the natural environment. Sucholigonucleotides may be obtained by a number of methods including, forexample, laboratory synthesis, restriction enzyme digestion or PCR. A“substantially purified” oligonucleotide is preferably greater than 50%pure, more preferably at least 75% pure, and most preferably at least95% pure.

As used herein, an oligonucleotide is “specific” for a nucleic acid ifthe oligonucleotide has at least 50% sequence identity with a portion ofthe nucleic acid when the oligonucleotide and the nucleic acid arealigned. An oligonucleotide that is specific for a nucleic acid is onethat, under the appropriate hybridization or washing conditions, iscapable of hybridizing to the target of interest and not substantiallyhybridizing to nucleic acids which are not of interest. Higher levels ofsequence identity are preferred and include at least 75%, at least 80%,at least 85%, at least 90%, at least 95% and more preferably at least98% sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well know in the art.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically and preferably conducted with oligonucleotides Nucleic acidhybridization techniques are well known in the art. See, e.g., Sambrook,et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the artunderstand how to estimate and adjust the stringency of hybridizationconditions such that sequences having at least a desired level ofcomplementarity will stably hybridize, while those having lowercomplementarity will not. For examples of hybridization conditions andparameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in MolecularBiology. John Wiley & Sons, Secaucus, N.J.

The terms “target nucleic acid” or “target sequence” as used hereinrefer to a sequence which includes an allele or mutation of interest tobe amplified and detected. Copies of the target sequence which aregenerated during the amplification reaction are referred to asamplification products, amplimers, or amplicons. Target nucleic acid maybe composed of segments of a chromosome, a complete gene with or withoutintergenic sequence, segments or portions of a gene with or withoutintergenic sequence, or sequence of nucleic acids which probes orprimers are designed. Target nucleic acids may include a wild-typesequences, a mutation, deletion or duplication, tandem repeat regions, agene of interest, a region of a gene of interest or any upstream ordownstream region thereof. Target nucleic acids may representalternative sequences or alleles of a particular gene. Target nucleicacids may be derived from genomic DNA, cDNA, or RNA. As used hereintarget nucleic acid may be DNA or RNA extracted from a cell or a nucleicacid copied or amplified therefrom.

“Genomic nucleic acid” or “genomic DNA” refers to some or all of the DNAfrom a chromosome. Genomic DNA may be intact or fragmented (e.g.,digested with restriction endonucleases by methods known in the art). Insome embodiments, genomic DNA may include sequence from all or a portionof a single gene or from multiple genes. In contrast, the term “totalgenomic nucleic acid” is used herein to refer to the full complement ofDNA contained in the genome. Methods of purifying DNA and/or RNA from avariety of samples are well-known in the art.

The term “flanking” as used herein means that a primer hybridizes to atarget nucleic acid adjoining a region of interest sought to beamplified on the target. The skilled artisan will understand thatpreferred primers are pairs of primers that hybridize 3′ from a regionof interest, one on each strand of a target double stranded DNAmolecule, such that nucleotides may be add to the 3′ end of the primerby a suitable DNA polymerase.

The term “complement” “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) refersto standard Watson/Crick pairing rules. The complement of a nucleic acidsequence such that the 5′ end of one sequence is paired with the 3′ endof the other, is in “antiparallel association.” For example, thesequence “5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-5′.”Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids described herein; these include, forexample, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementary need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. A complement sequence can also be asequence of RNA complementary to the DNA sequence or its complementsequence, and can also be a cDNA.

The term “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences can comprise a contiguous sequenceof bases that do not hybridize to a target sequence, positioned 3′ or 5′to a contiguous sequence of bases that hybridize under stringenthybridization conditions to a target sequence.

The term “coding sequence” as used herein means a sequence of a nucleicacid or its complement, or a part thereof, that can be transcribedand/or translated to produce the mRNA for and/or the polypeptide or afragment thereof. Coding sequences include exons in a genomic DNA orimmature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the encodingsequence can be deduced from there.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be either DNA or RNA.The sequences amplified in this manner form an “amplicon.” While theexemplary methods described hereinafter relate to amplification usingthe polymerase chain reaction (PCR), numerous other methods are known inthe art for amplification of nucleic acids (e.g., isothermal methods,rolling circle methods, etc.). The skilled artisan will understand thatthese other methods may be used either in place of, or together with,PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCRProtocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990,pp 13-20; Wharam, et al., Nucleic Acids Res. 2001 Jun. 1;29(11):E54-E54; Hafner, et al., Biotechniques 2001 April; 30(4):852-6,858, 860 passim; Zhong, et al., Biotechniques 2001 April; 30(4):852-6,858, 860.

The term “multiplex PCR” as used herein refers to simultaneousamplification of two or more products which are each primed using adistinct primer pair.

As used herein, a “primer” for amplification is an oligonucleotide thatspecifically anneals to a target nucleotide sequence and leads toaddition of nucleotides to the 3′ end of the primer in the presence of aDNA or RNA polymerase. The 3′ nucleotide of the primer should generallybe identical to the target sequence at a corresponding nucleotideposition for optimal expression and amplification. The term “primer” asused herein includes all forms of primers that may be synthesizedincluding peptide nucleic acid primers, locked nucleic acid primers,phosphorothioate modified primers, labeled primers, and the like.

“Sense strand” means the strand of double-stranded DNA (dsDNA) thatincludes at least a portion of a coding sequence of a functionalprotein. “Anti-sense strand” means the strand of dsDNA that is thereverse complement of the sense strand.

As used herein, a “forward primer” is a primer that anneals to theanti-sense strand of dsDNA. A “reverse primer” anneals to thesense-strand of dsDNA.

As used herein, sequences that have “high sequence identity” haveidentical nucleotides at least at about 50% of aligned nucleotidepositions, preferably at least at about 58% of aligned nucleotidepositions, and more preferably at least at about 76% of alignednucleotide positions.

As used herein “TaqMan® PCR detection system” refers to a method forreal time PCR. In this method, a TaqMan® probe which hybridizes to thenucleic acid segment amplified is included in the PCR reaction mix. TheTaqMan® probe comprises a donor and a quencher fluorophore on either endof the probe and in close enough proximity to each other so that thefluorescence of the donor is taken up by the quencher. However, when theprobe hybridizes to the amplified segment, the 5′-exonuclease activityof the Taq polymerase cleaves the probe thereby allowing the donorfluorophore to emit fluorescence which can be detected.

As used herein, “about” means plus or minus 10%.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic diagram of primer placement for mutant specific PCR ofa deletion mutation exemplified by E746_A750del in the EGFR gene. Thedeleted sequence is shown as a dashed line in the EGFR wild-type DNA.Horizontal arrows indicate primer placement for forward and reverseprimers.

FIG. 2A. Nucleotide sequence of a portion of the sequence coding for thewild-type form of the EGFR (SEQ ID NO:22). The two segments ofhighlighted unbolded text together represent the sequence for a forwardmutation specific PCR primer (SEQ ID NO:1) specific for the E746_A750delmutant EGFR gene. Only a portion of the forward mutation specific primeris complementary to a contiguous segment of the wild-type EGFR gene.Highlighted bolded text indicates sequence for a reverse PCR primer (SEQID NO:2). The 15 bp region that is deleted is located between the twoportions of the forward primer in the E746_A750del mutant EGFR gene.Boxed TTAA regions indicate MseI restriction sites.

FIG. 2B. Nucleotide sequence of a portion of the coding region of theE746_A750del mutant of EGFR (SEQ ID NO:23. Highlighted unbolded textindicates sequence of a forward mutation specific PCR primer (SEQ IDNO:1) specific for the E746_A750del mutant EGFR gene. Highlighted boldedtext indicates sequence for a reverse PCR primer (SEQ ID NO:2). BoxedTTAA regions indicate MseI restriction sites.

FIG. 3. Schematic diagram of primer placement for mutant specific PCR ofan insertion mutation exemplified by the exon 16 mutation in the APCgene. Horizontal arrows indicate primer placement for forward andreverse primers. The gray region represents the inserted sequence. Whitestars indicate MnlI restriction sites.

FIG. 4. Nucleotide sequence of a portion of APC gene showing the exon 16insertion sequence in unbolded text (SEQ ID NO:24). Highlighted boldedtext indicates the sequence for a forward PCR primer (SEQ ID NO:3) andhighlighted unbolded text indicates the sequence of a reverse PCR primer(SEQ ID NO:4). Boxed GAGG and CCTC regions indicate MnlI restrictionsites.

FIG. 5. Schematic diagram of alternative primer placement for mutantspecific PCR of an insertion mutation exemplified by the exon 16mutation in the APC gene. Horizontal arrows indicate primer placementfor forward and reverse primers. The gray region represents the insertedsequence. White stars indicate MnlI restriction sites.

FIG. 6. Nucleotide sequence of a portion of APC gene showing the exon 16insertion sequence in unbolded text (SEQ ID NO:25). Highlighted boldedtext indicates the sequence for a forward PCR primer (SEQ ID NO:5).Highlighted unbolded text indicates the sequence of a reverse PCR primer(SEQ ID NO:6). Boxed GAGG and CCTC regions indicate MnlI restrictionsites.

FIG. 7. Schematic diagram of primer placement for mutant specific PCR ofa substitution mutation exemplified by point mutation L858R in the EGFRgene. Horizontal arrows indicate primer placement for forward andreverse primers. The boxed sequence indicates the EaeI restriction site.The bolded “G” base pair represents the substituted base (SEQ ID NO:26and SEQ ID NO:27, respectively in order of appearance).

FIG. 8. Nucleotide sequences of portions of wild-type (SEQ ID NO:28) andmutant EGFR genes (SEQ ID NO:29). Highlighted unbolded text indicatesthe sequence for a forward mutation specific PCR primer (SEQ ID NO:7)specific for the L858R mutant EGFR gene. Only a portion of the forwardmutation specific primer is complementary to a contiguous segment of thewild-type EGFR gene. Highlighted bolded text indicates the sequence fora reverse PCR primer (SEQ ID NO:8). The bolded boxed base pair “T”indicates where the point mutation occurs in the L858R mutant EGFR gene.The bolded “G” in the forward mutation specific primer is the locationof the locked nucleic acid. Highlighted unbolded text in the wild-typeEGFR gene sequence indicates where the forward mutation specific primerwould hybridize, or base pair. Boxed TTAA regions indicate MseIrestriction sites. Boxed YGGCCR region, where Y=C or T; and R=A or G,indicates an EaeI restriction site.

FIG. 9. Schematic diagram of primer placement for mutant specific PCR ofa translocation mutation exemplified by the TMPRSS2:ERG fusiontranscript. Horizontal arrows indicate primer placement for forward andreverse primers. White stars indicate FatI restriction sites.

FIG. 10. Nucleotide sequences of portions of wild-type TMPRSS2 (SEQ IDNO:30) and ERG (SEQ ID NO:31) and mutant fusion (SEQ ID NO:32) gene,TMPRSS2:ERG. Highlighted bolded text indicates the sequence for aforward mutation specific PCR primer (SEQ ID NO:9). Highlighted unboldedtext indicates the sequence for a reverse mutation specific PCR primer(SEQ ID NO:10). Underlined regions in the wild-type sequences correspondto the depicted portion of the resulting fusion gene. Boxed CATG regionsindicate FatI restriction sites.

FIG. 11. Schematic diagram of primer placement for mutant specific PCRof a translocation mutation exemplified by the TMPRSS2:ETV1 fusiontranscript. Horizontal arrows indicate primer placement for forward andreverse primers. White stars indicate FatI and HpyCH4V restrictionsites.

FIG. 12. Nucleotide sequences of portions of wild-type TMPRSS2 (SEQ IDNO:33) and ETVI (SEQ ID NO:34) and mutant fusion (SEQ ID NO:35) gene,TMPRSS2:ETV1. Highlighted bolded text indicates sequence for a forwardmutation specific PCR primer (SEQ ID NO:11). Highlighted unbolded textindicates sequence for a reverse mutation specific PCR primer (SEQ IDNO:12). Underlined regions in the wild-type sequences correspond to thedepicted portion of the resulting fusion gene. Boxed CATG and TGCAregions indicate HpyCH4V restriction sites.

FIG. 13. Graphical depiction of results of sensitivity comparison assaybetween Sanders method and Asano method for detection of theE746_A750del mutation in EGFR.

FIG. 14. Comparison of eight DNA extraction methods. Columns indicatethe mean percent recovery of six plasma samples as described in Example5.

FIG. 15. Evaluation of detection between Agencourt Genfind™, RocheCobas® and phenol:chloroform extraction using Eppendorf Phase Lock Gels®methods using spiked plasma samples as described in Example 5. Columnsrepresent the mean peak intensity of E746_A750del PCR product from thesix samples tested for each method obtained from an ABI 3100 GeneticAnalyzer.

FIG. 16. Evaluation of nucleic acid spiking conditions as described inExample 5. Six nucleic acid carrier conditions include 1) no carrier, 2)no carrier plus 100 ng of normal DNA, 3) 1 μg of RNA carrier, 4) 1 μg ofRNA carrier plus 100 ng of normal DNA, 5) 395 ng of normal DNA as acarrier, and 6) 778 ng of normal DNA as a carrier. Columns represent themean peak intensity of E746_A750del PCR product.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods fordetermining whether a sample contains target nucleic acid. The methodsoutlined for detection of specific deletion mutations, insertionmutations, point mutations and translocation mutations can be applied toany biomarker in proximity to a restriction digestion recognition site,preferably a restriction digestion recognition site is disrupted by oneor more mutations. A blueprint of the primer designs is depicted belowbut primer sequences will vary to fit the mutation to be detected.Restriction enzyme digestion sites will also depend on the sequence ofthe non-target sequence as compared to the target nucleic acid or fusiontranscript but can follow the formats below. The frequency of variousrestriction sites found in DNA virtually ensures that a site unique tothe non-target DNA of interest can be found for any target detectionassay, thus these methodologies are applicable to a wide array of cancerbiomarkers.

Primers

For the methods provided herein, a single primer could be used fordetection, for example as in single nucleotide primer extension, or asecond primer can be used which can be upstream or downstream of themutation specific primer. One or more of the primers used may bemutation specific primers. Preferably, the mutation specific primercontains wild-type sequence, more preferably at least about 3-40consecutive nucleotides of wild-type sequence.

Fragmentation

Fragmentation is preferably achieved by restriction enzyme treatment orone of other methods of fragmentation well known in the art. In order toreduce the likelihood of mis-priming or inability of the decreasedability for the primer to find a low copy target sequence amongnon-target sequences, a restriction enzyme recognition site ispreferably present in the deleted sequence. Restriction digestiontreatment prior to amplification will then cleave non-target sequences.Preferably, the mutation destroys a restriction enzyme recognition sitesuch that the wild-type sequence will be digested, but the mutantsequence no longer contains the recognition site.

One of skill in the art would recognize that a restriction enzymefragmentation method can be modified by using a restriction enzyme thatcuts at a particular frequency or a particular site, or by usingmultiple restriction enzymes. The choice of enzyme or enzymecombinations is chosen to suit the target of interest in an assay.Enzymes for fragmentation can be chosen by using a restriction enzymemap of the region of interest. Such maps can be readily generated bysoftware programs well-known to those of skill in the art.

Chemical fragmentation may include degradation by a nuclease such asDNase or RNase which generate fragments having 3′-OH, 5′-OH,3′-phosphate and 5′-phosphate ends; depurination or depyrimidation withacid; the use of restriction enzymes; intron-encoded endonucleases;DNA-based cleavage methods, such as triplex and hybrid formationmethods, that rely on the specific hybridization of a nucleic acidsegment to localize a cleavage agent to a specific location in thenucleic acid molecule; or other enzymes or compounds which cleave DNA atknown or unknown locations (see, for example, U.S. Pat. No. 6,495,320).It is possible to depurinate or depyrimidinate the DNA, which is thenfragmented in the presence of a base (i.e., “β-elimination”) DNA can befragmented by oxidation, alkylation or free radical addition mechanisms.Metal cations, which are often combined with organic molecules which mayfunction as chemical catalysts, for example imidazole, are used forfragmenting RNA. This fragmentation is preferably carried out in analkaline medium and generates fragments having 3′-phosphate ends.Chemical catalysts that may be used for nucleic acid fragmentationinclude MOPS, HEPES, PIPES, and bioorganic polyamines, such as spermine,spermidine and putrescine (Bibille et al., 27 Nucleic Acids Res.3931-3937 (1999)).

Different nucleic acid fragmentation techniques have been described, forexample, in Trawick et al., 98 Chem Rev. 939-960 (1998), Oivanen at al.,1998, 98 Chem Rev. 961-990 (1998) and Laayoon, et al. U.S. Pat. No.6,902,891. A method for fragmenting and labeling RNA is described inWO88/04300A1, in which fragmentation is carried out using RNA whichpossesses enzymatic properties (ribozymes).

Physical fragmentation methods may involve subjecting the DNA to a highshear rate. High shear rates may be produced, for example, by moving DNAthrough a chamber or channel with pits or spikes, or forcing the DNAsample through a restricted size flow passage, e.g., an aperture havinga cross sectional dimension in the micron or submicron scale. Otherphysical methods include sonication and nebulization. Combinations ofphysical and chemical fragmentation methods may likewise be employedsuch as fragmentation by heat and ion-mediated hydrolysis. See forexample, Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3rdEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

Preferential cleavage can be achieved my other methods known in the artsuch as the Maxam-Gilbert method. This method involves degrading DNA ata specific base using chemical reagents. A. M. Maxim et al., 65(1) Meth.in Enzym. 499-560 (1980). In general, this method starts with endlabeled DNA and cleaves by base specific reagents. For example withguanine bases (the same principle applies to all four bases), DNA ofinterest is end-labeled (can be 5′- or 3′-end labeling). Then one kindof base is modified, for example with dimethyl sulfate (DMS) tomethylate guanines. Conditions can be adjusted to achieve variousfrequencies of methylation. Following methylation. a reagent such aspiperidine is added which causes loss of a methylated base and thenbreaks the DNA backbone at the site of the lost base (the apurinicsite).

Deletion Mutations

In one approach, a mutation specific primer is designed for detecting adeletion mutation. Mutation specific primer can be designed to span thedeleted region such that the primer contains wild-type sequence thatlies 5′ and 3′ of the deleted region or the complement thereof. Thus,the mutation specific primer cannot bind to the wild-type sequence andcannot produce an amplicon.

Oligonucleotide primers may be designed for amplifying regions ofmutated nucleic acid. In one approach, a primer pair is designed fordetecting a deletion mutation. In one embodiment, the primer pair isdesigned to hybridize to a specified segment of the EGFR gene. Thesequence of exemplary oligo primers are shown as highlighted regions inFIGS. 2A and 2B (SEQ ID NOs:1 and 2). Exemplary primer pairs foramplifying a region of the EGFR sequence for the E746_A750del mutationuse a forward primer (mutation specific primer) with SEQ ID NO:1(5′-CCCGTCGCTATCAAAACATC-3′) and a reverse primer with SEQ ID NO:2(5′-ATGTGGAGATGAGCAGGGTCT-3′). In this example, the mutation specificprimer spans sequence that is deleted in the mutated sequence. Thus, theprimer cannot anneal to the wild-type sequence due to the presence ofthe 15 base pairs. Preferably, the primers with SEQ ID NOs:1 and 2 areeach or both used in conjunction with a restriction enzyme digestiontreatment with MseI which has a recognition site of TTAA. The mutationspecific primer in that example lies 5′ and 3′ of the 15 bp deletedregion, it cannot bind to the wild-type sequence, thus making the primermutation specific for the deletion mutation. FIGS. 1 and 2 illustratethis example of detecting the E746_A750del mutation in the EGFR gene.

Insertion Mutations

In one approach, a mutation specific primer is designed for detecting aninsertion mutation. A mutation specific primer can be designed to spanall or a portion of the inserted region such that the primer includesall or a part of the inserted region. A primer could be designed to spanthe either or both junctions of the inserted sequence, for example, theprimer sequence would include a portion of wild-type sequence that isadjacent to the inserted sequence or the complement thereof. Thus, themutation specific primer is not complementary to the wild-type sequenceand cannot produce an amplicon.

Preferably, the insertion destroys a restriction enzyme recognition sitesuch that the wild-type sequence will be digested, but the mutantsequence no longer contains the recognition site. Restriction digestiontreatment prior to amplification will then cleave non-target sequences.Restriction digestion can also enhance sensitivity by cleaving awaysequence surrounding target nucleic acid and facilitate amplification.

In one embodiment, a primer pair is designed to detect the 758 base pairinsertion in exon 16 insertion in the APC gene. The sequence ofexemplary oligo primers are shown as highlighted regions in FIG. 4 (SEQID NOs:3 and 4). Exemplary primer pairs for amplifying a region of theAPC sequence for an exon 16 (Miki, et al., 52(3) Cancer Research 643-645(1992)) insertion mutation use a forward primer (mutation specificprimer) with SEQ ID NO:3 (5′-CTTCCACAATGGTTGAACTAG-3′) and a reverseprimer (mutation specific primer) with SEQ ID NO:4(5′-CATCCATGTCCCTACAAAGG-3′). In this example, both forward and reverseprimers are mutation specific because they lie within the insertionsequence. Preferably, the primers with SEQ ID NOs:3 and 4 are each orboth used in conjunction with a restriction enzyme digestion treatmentwith MnlI which has a recognition site of CCTC.

The mutation specific primer in that example lies within the insertedregion. Because the primers lie within the insertion sequence, noamplification will occur unless the insertion is present. In addition,there are MnlI restriction sites upstream and downstream of the desiredamplification product which will facilitate amplification subsequent todigestion by removing surrounding sequence. FIGS. 3 and 4 illustratethis example of detecting the exon 16 insertion mutation in the APCgene.

In another embodiment, a primer pair is designed to detect the 758 basepair insertion in exon 16 insertion in the APC gene. The sequence ofexemplary oligo primers are shown as highlighted regions in FIG. 6 (SEQID NOs:5 and 6). Exemplary primer pairs for amplifying a region of theAPC sequence for an exon 16 insertion mutation use a forward primer(mutation specific primer) with SEQ ID NO:5(5′-GAGCCATTTATACAGAAAGATG-3′) and a reverse primer (mutation specificprimer) with SEQ ID NO:6 (5′-GAAATACCATTTGACCCAGC-3′). In this example,the forward primer lies outside the insertion sequence and reverseprimer lies inside the insertion sequence. Both are mutation specificbecause an amplicon will not be produced in the absence of the insertionsequence. Preferably, the primers with SEQ ID NOs:5 and 6 are each orboth used in conjunction with a restriction enzyme digestion treatmentwith MnlI which has a recognition site of CCTC.

One primer is upstream of the insertion site and the second is withinthe insertion sequence. In addition, there are MnlI restriction sitesless than 20 bases upstream and downstream of the desired amplificationproduct which will facilitate amplification of the target nucleic acidby removing surrounding sequence. The wild-type APC sequence is heavilytargeted by MnlI restriction enzyme with a number of sites immediatelydownstream of the forward primer. The insertion sequence in the APC exon16 insertion mutant contains a region devoid of MnlI restriction sitesthat is used as the template for PCR. Digestion with MnlI prior to PCReliminates any linear amplification that may occur by forward primerbinding to the wild type APC gene. Fluorescent PCR can be performedusing one forward primer that binds to the APC gene just before theinsertion and one insertion specific reverse primer (unlabeled forward,FAM labeled reverse) designed to specifically recognize the insertionsequence in the region not containing MnlI restriction sites. FIGS. 5and 6 illustrate this example of detecting the exon 16 insertionmutation in the APC gene.

Substitution Mutations

In one approach, a mutation specific primer is designed for detectingone or more substitution mutations. A mutation specific primer can bedesigned to include one or more substitutions. In a preferredembodiment, the 3′ nucleotide of the primer can be designed such that itcontains the mutated base pair and does not bind, hybridize, or basepair, in the wild-type gene and thus cannot elongate. In anotherpreferred embodiment, the mutated base pair is located at the −1position at the 3′-end of a mutation specific primer (i.e., thepenultimate base).

Preferably, the one or more substitutions destroys a restriction enzymerecognition site such that the wild-type sequence will be digested, butthe mutant sequence no longer contains the recognition site. Restrictiondigestion treatment prior to amplification will then cleave non-targetsequences.

In further preferred embodiments, the mutated base pair in the mutationspecific primer is a locked nucleic acid (LNA). The locked nucleic acidprovides increased specificity by increasing the melting temperature ofthe of a primer containing the substitution base. This allows for theuse of an increased annealing temperature during amplification whichdecreases amplification of wild type sequences.

In one embodiment, a primer pair is designed to detect the L858Rmutation in the EGFR gene. The sequence of exemplary oligo primers areshown as highlighted regions in FIG. 8 (SEQ ID NOs:7 and 8). Exemplaryprimer pairs for amplifying a region of the EGFR sequence for the L858Rmutation use a forward primer (mutation specific primer) with SEQ IDNO:7 (5′-TCACAGATTTTGGGCGG-3′) and a reverse primer with SEQ ID NO:8(5′-CCTGGTGTCAGGAAAATGCT-3′). In this example, the mutation specificprimer contains the mutated sequence at the terminal base. Thus, it willnot properly anneal to the wild-type sequence because the last base isnot complementary. Preferably, the primers with SEQ ID NOs:7 and 8 areeach or both used in conjunction with a restriction enzyme digestiontreatment with EaeI which has a recognition site of YGGCCR, where Y=C orT and R=A or G. As shown in FIG. 8, the boxed MseI restriction sites,TTAA, illustrate that a simultaneous reaction, such as a multiplex PCRreaction, can be used to detect either or both the E746_A750del andL858R mutations in the same reaction. Digestion with both MseI and EaeIdoes not disrupt the L858R sequence of interest.

The mutation specific primer in that example includes the mutated basepair sequence, a G, at its 3′ end. Because the primer is notcomplementary to the wild-type sequence, which contains a T, elongationwill not occur. The EaeI cut site allows cleavage of the wild-type EGFRgene but is destroyed by the T-*G conversion. Thus, when the L858R EGFRmutant is present, the recognition site is no longer present and can nolonger be digested by EaeI. FIGS. 7 and 8 illustrate this example ofdetecting the L858R mutation in the EGFR gene.

Translocation Mutations

In one approach, a mutation specific primer is designed for detectingone or more translocation mutations. A mutation specific primer can bedesigned to span the junction of the translocation or the complementthereof. A primer pair could be designed to so that one primer isupstream of the translocation junction and the second is downstream ofthe junction. Thus, when the primer pair is used on wild-type sequence,no amplification products will be produced because the locations of theprimers relative to each other are cannot be amplified. However, whenthe translocation is present, the primers are in close enough proximityof each other such that an amplification product can be produced. Forexample, the primer can be designed to include a portion of the firstgene and a portion of the second gene, where the genes are located ondifferent chromosomes in wild-type form but are adjacent to one anotherin the mutated form.

Preferably, one or more translocations destroys a restriction enzymerecognition site such that the wild-type sequence will be digested, butthe mutant sequence no longer contains the recognition site. Restrictiondigestion treatment prior to amplification will then cleave non-targetsequences.

In one embodiment, a primer pair is designed to detect the TMPRSS2:ERGor translocation mutation of the TMPRSS2 and ERG genes. The sequence ofexemplary oligo primers are shown as higlighted regions in FIG. 10 (SEQID NOs:9 and 10). Exemplary primer pairs for amplifying a region of theTMPRSS2 and ERG sequences for the TMPRSS2:ERG translocation mutation usea forward primer (mutation specific primer) with SEQ ID NO:9(5′-CGAGCTAAGCAGGAGGCGG-3′) and a reverse primer (mutation specificprimer) with SEQ ID NO:10 (5′-GTCCATAGTCGCTGGAGGAG-3′). In this example,while both primers anneal to wild-type sequences, they are mutationspecific when used in conjunction with each other because they will notproduce an amplification product unless the translocation is present inthe nucleic acid sample. Preferably, the primers with SEQ ID NOs:9 and10 are each or both used in conjunction with a restriction enzymedigestion treatment with FatI which has a recognition site of CATG.

In another embodiment, a primer pair is designed to detect theTMPRSS2:ETV1 translocation mutations of the TMPRSS2 and ETV1 genes. Thesequence of exemplary oligo primers are shown as highlighted regions inFIG. 12 (SEQ ID NOs:11 and 12). Exemplary primer pairs for amplifying aregion of the TMPRSS2 and ERG sequences for the TMPRSS2:ETV1translocation mutation use a forward primer (mutation specific primer)with SEQ ID NO:11 (5′-CGAGCTAAGCAGGAGGCGG-3′) and a reverse primer(mutation specific primer) with SEQ ID NO:12(5′-ACTTTCAGCCTGATAGTCTGG-3′). In this example, while both primersanneal to wild-type sequences, they are mutation specific when used inconjunction with each other because they will not produce anamplification product unless the translocation is present in the nucleicacid sample. Preferably, the primers with SEQ ID NOs:11 and 12 are eachor both used in conjunction with a restriction enzyme digestiontreatment with HpyCH4VI which has a recognition site of TGCA.

In these embodiments, the Fat I and HpyCH4V cut sites allow cleavage ofthe wild-type TMPRSS2, ERG, and ETV1 in the regions that are absent inthe fusion transcripts, essentially “decontaminating” the sample ofwild-type TMPRSS2 and ERG or ETV1 translocations. Because the forwardand reverse primer sequences are only both present in the fusiontranscripts, only the nucleic acids representing a fusion transcriptwill yield PCR products. FIGS. 9 and 10 illustrate this example ofdetecting the TMPRSS2:ERG translocation mutation.

Sample Preparation

The method may be performed using any sample containing nucleic acid.Samples may be obtained by standard procedures and may be usedimmediately or stored (e.g., the sample may be frozen between about −15°C. to about −100° C.) for later use. Samples may be obtained frompatients suspected of having a mutated nucleic acid sequence, forexample from a tumor cell or cancer cells. The presence of mutatednucleic acids in a sample can be determined by amplifying cancer markerregions. Thus, any liquid or solid material believed to contain cancermarker nucleic acids can be an appropriate sample. Preferred sampletissues include plasma, blood, bone marrow, body fluids, cerebrospinalfluid, urine and others. Heparin is known to inhibit PCR (Beutler, etal. BioTechniques 9:166, 1990), so samples containing heparin are notideal for the uses contemplated herein. Nucleic acid extractiontechniques that remove heparin are known in the art. These techniquesmay be used to remove heparin from samples to make the samples moresuitable for amplification.

The sample may be processed to release or otherwise make available anucleic acid for detection as described herein. Such processing mayinclude steps of nucleic acid manipulation, e.g., preparing a cDNA byreverse transcription of RNA from the biological sample. Thus, thenucleic acid to be amplified by the methods of the invention may begenomic DNA, cDNA, single stranded DNA or mRNA.

Oligonucleotides

Oligonucleotide primers may be approximately 15-100 nucleotides inlength. Of the specific oligonucleotides provided herein, additionalvariations of the primers comprise all or a portion of the SEQ IDsdescribed herein. Other preferred oligonucleotide primers include anoligonucleotide sequence that hybridizes to the complement of a 15-100nucleotide sequence that comprises the complement of all or a portion ofthe SEQ IDs described herein. Such oligonucleotides may be substantiallypurified.

Amplification of Nucleic Acids

Nucleic acid samples or isolated nucleic acids may be amplified byvarious methods known to the skilled artisan. Preferably, PCR is used toamplify mutated nucleic acids of interest. In this method, two or moreoligonucleotide primers that flank or include, and anneal to oppositestrands of a nucleic acid of interest are repetitively annealed to theircomplementary sequences, extended by a DNA polymerase (e.g., AmpliTaqGold polymerase), and heat denatured, resulting in exponentialamplification of the target nucleic acid sequences. Cycling parameterscan be varied, depending on the length of nucleic acids to be extended.The skilled artisan is capable of designing and preparing primers thatare appropriate for amplifying a target sequence in view of thisdisclosure. The length of the amplification primers for use in thepresent invention depends on several factors including the nucleotidesequence identity and the temperature at which these nucleic acids arehybridized or used during in vitro nucleic acid amplification. Theconsiderations necessary to determine a preferred length for anamplification primer of a particular sequence identity are well known tothe person of ordinary skill. For example, the length of a short nucleicacid or oligonucleotide can relate to its hybridization specificity orselectivity.

Assay controls may be used in the assay for detecting a mutated nucleicacid sequence. An internal positive amplification control (IPC) can beincluded in the sample, utilizing oligonucleotide primers and/or probes.

Detection of Amplified Nucleic Acids

Amplification of nucleic acids can be detected by any of a number ofmethods well-known in the art such as gel electrophoresis, columnchromatography, hybridization with a probe, or sequencing.

In one approach, sequences from two or more regions of interest areamplified in the same reaction vessel. In this case, the amplicon(s)could be detected by first size-separating the amplicons then detectionof the size-separated amplicons. The separation of amplicons ofdifferent sizes can be accomplished by, for example, gelelectrophoresis, column chromatography, or capillary electrophoresis.These and other separation methods are well-known in the art. In oneexample, amplicons of about 10 to about 150 base pairs whose sizesdiffer by 10 or more base pairs can be separated, for example, on a 4%to 5% agarose gel, (a 2% to 3% agarose gel for about 150 to about 300base pair amplicons) or a 6% to 10% polyacrylamide gel. The separatednucleic acids can then be stained with a dye such as ethidium bromideand the size of the resulting stained band or bands can be compared to astandard DNA ladder.

In another embodiment, two or more regions of interest are amplified inseparate reaction vessels. If the amplification is specific, that is,one primer pair amplifies for one region of interest but not the other,detection of amplification is sufficient to distinguish between the twotypes—size separation would not be required.

In some embodiments, amplified nucleic acids are detected byhybridization with a mutation-specific probe. Probe oligonucleotides,complementary to a portion of the amplified target sequence may be usedto detect amplified fragments. Amplified nucleic acids for each of thetarget sequences may be detected simultaneously (i.e., in the samereaction vessel) or individually (i.e., in separate reaction vessels).In preferred embodiments, the amplified DNA is detected simultaneously,using two distinguishably-labeled, gene-specific oligonucleotide probes,one which hybridizes to the first target sequence and one whichhybridizes to the second target sequence.

The probe may be detectably labeled by methods known in the art. Usefullabels include, e.g., fluorescent dyes (e.g., Cy5®, Cy3®, FITC,rhodamine, lanthamide phosphors, Texas red), 32P, 35S, 3H, 14C, 125I,131I, electron-dense reagents (e.g., gold), enzymes, e.g., as commonlyused in an ELISA (e.g., horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), colorimetric labels (e.g., colloidalgold), magnetic labels (e.g., Dynabeads™), biotin, dioxigenin, orhaptens and proteins for which antisera or monoclonal antibodies areavailable. Other labels include ligands or oligonucleotides capable offorming a complex with the corresponding receptor or oligonucleotidecomplement, respectively. The label can be directly incorporated intothe nucleic acid to be detected, or it can be attached to a probe (e.g.,an oligonucleotide) or antibody that hybridizes or binds to the nucleicacid to be detected.

A probe oligonucleotide, complementary to the amplified region ofnucleic acid, is used to detect the amplification of mutated nucleicacids. The probe may be detectably labeled by methods known in the art.The binding of a probe to the amplified region of the mutated nucleicacid may be determined by hybridization as is well known in the art.Hybridization may be detected in real time or in non-real time.

One general method for real time PCR uses fluorescent probes such as theTaqMan® probes, molecular beacons and scorpions. Real-timereverse-transcriptase (RT) PCR quantitates the initial amount of thetemplate with more specificity, sensitivity and reproducibility, thanother forms of quantitative reverse transcriptase PCR, which detect theamount of final amplified product. Real-time RT-PCR does not detect thesize of the amplicon. The probes employed in TaqMan® and molecularbeacon technologies are based on the principle of fluorescence quenchingand involve a donor fluorophore and a quenching moiety.

In a preferred embodiment, the detectable label is a fluorophore. Theterm “fluorophore” as used herein refers to a molecule that absorbslight at a particular wavelength (excitation frequency) and subsequentlyemits light of a longer wavelength (emission frequency). The term “donorfluorophore” as used herein means a fluorophore that, when in closeproximity to a quencher moiety, donates or transfers emission energy tothe quencher. As a result of donating energy to the quencher moiety, thedonor fluorophore will itself emit less light at a particular emissionfrequency that it would have in the absence of a closely positionedquencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.In the latter case, the quencher is considered to be an acceptorfluorophore. The quenching moiety can act via proximal (i.e.,collisional) quenching or by Förster or fluorescence resonance energytransfer (“FRET”). Quenching by FRET is generally used in TaqMan® probeswhile proximal quenching is used in molecular beacon and scorpion typeprobes.

In proximal quenching (a.k.a. “contact” or “collisional” quenching), thedonor is in close proximity to the quencher moiety such that energy ofthe donor is transferred to the quencher, which dissipates the energy asheat as opposed to a fluorescence emission. In FRET quenching, the donorfluorophore transfers its energy to a quencher which releases the energyas fluorescence at a higher wavelength. Proximal quenching requires veryclose positioning of the donor and quencher moiety, while FRETquenching, also distance related, occurs over a greater distance(generally 1-10 nm, the energy transfer depending on R-6, where R is thedistance between the donor and the acceptor). Thus, when FRET quenchingis involved, the quenching moiety is an acceptor fluorophore that has anexcitation frequency spectrum that overlaps with the donor emissionfrequency spectrum. When quenching by FRET is employed, the assay maydetect an increase in donor fluorophore fluorescence resulting fromincreased distance between the donor and the quencher (acceptorfluorophore) or a decrease in acceptor fluorophore emission resultingfrom increased distance between the donor and the quencher (acceptorfluorophore).

Suitable fluorescent moieties include the following fluorophores knownin the art:

-   4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid-   acridine and derivatives:    -   acridine    -   acridine isothiocyanate-   Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor®    555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular    Probes)-   5-(2′-aminoethy)aminonaphthalene-1-sulfonic acid (EDANS)-   4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate    (Lucifer Yellow VS)-   N-(4-anilino-1-naphthyl)maleimide-   anthranilamide-   Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies)-   BODIPY® R-6G, BOPIPY® 530/550, BODIPY® FL-   Brilliant Yellow-   coumarin and derivatives:    -   coumarin    -   7-amino-4-methylcoumarin (AMC, Coumarin 120)-   7-amino-4-trifluoromethylcouluarin (Coumarin 151)-   Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®-   cyanosine-   4′,6-diaminidino-2-phenylindole (DAPI)-   5′, 5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)-   7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin-   diethylenetriamine pentaacetate-   4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid-   4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid-   5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl    chloride)-   4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)-   4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC)-   Eclipse™ (Epoch Biosciences Inc.)-   eosin and derivatives:    -   eosin    -   eosin isothiocyanate-   erythrosin and derivatives:    -   erythrosin B    -   erythrosin isothiocyanate-   ethidium-   fluorescein and derivatives:    -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)    -   2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate (FITC)    -   hexachloro-6-carboxyfluorescein (HEX)    -   QFITC (XRITC)    -   tetrachlorofluorescein (TET)-   fluorescamine-   IR144-   IR1446-   Malachite Green isothiocyanate-   4-methylumbelliferone-   ortho cresolphthalein-   nitrotyrosine-   pararosaniline-   Phenol Red-   B-phycoerythrin, R-phycoerythrin-   o-phthaldialdehyde-   Oregon Green®-   propidium iodide-   pyrene and derivatives:    -   pyrene    -   pyrene butyrate    -   succinimidyl 1-pyrene butyrate-   QSY® 7, QSY® 9, QSY® 21, QSY® 35 (Molecular Probes)-   Reactive Red 4 (Cibacron® Brilliant Red 3B-A)-   rhodamine and derivatives:    -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride    -   rhodamine (Rhod)    -   rhodamine B    -   rhodamine 123    -   rhodamine green    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)-   N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)-   tetramethyl rhodamine-   tetramethyl rhodamine isothiocyanate (TRITC)-   riboflavin-   rosolic acid-   terbium chelate derivatives

Other fluorescent nucleotide analogs can be used, see, e.g., Jameson,278 Meth. Enzymol. 363-390 (1997); Zhu, 22 Nucl. Acids Res. 3418-3422(1994). U.S. Pat. Nos. 5,652,099 and 6,268,132 also describe nucleosideanalogs for incorporation into nucleic acids, e.g., DNA and/or RNA, oroligonucleotides, via either enzymatic or chemical synthesis to producefluorescent oligonucleotides. U.S. Pat. No. 5,135,717 describesphthalocyanine and tetrabenztriazaporphyrin reagents for use asfluorescent labels.

The detectable label can be incorporated into, associated with orconjugated to a nucleic acid. Label can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance or impact on otheruseful or desired properties. See, e.g., Mansfield, 9 Mol. Cell. Probes145-156 (1995).

Detectable labels can be incorporated into nucleic acids by covalent ornon-covalent means, e.g., by transcription, such as by random-primerlabeling using Klenow polymerase, or nick translation, or amplification,or equivalent as is known in the art. For example, a nucleotide base isconjugated to a detectable moiety, such as a fluorescent dye, e.g., Cy3®or Cy50 and then incorporated into genomic nucleic acids during nucleicacid synthesis or amplification. Nucleic acids can thereby be labeledwhen synthesized using Cy3®- or Cy5®-dCTP conjugates mixed withunlabeled dCTP.

Nucleic acid probes can be labeled by using PCR or nick translation inthe presence of labeled precursor nucleotides, for example, modifiednucleotides synthesized by coupling allylamine-dUTP to thesuccinimidyl-ester derivatives of the fluorescent dyes or haptens (suchas biotin or digoxigenin) can be used; this method allows custompreparation of most common fluorescent nucleotides, see, e.g.,Henegariu, 18 Nat. Biotechnol. 345-348 (2000).

Nucleic acid probes may be labeled by non-covalent means known in theart. For example, Kreatech Biotechnology's Universal Linkage System®(ULS®) provides a non-enzymatic labeling technology, wherein a platinumgroup forms a co-ordinative bond with DNA, RNA or nucleotides by bindingto the N7 position of guanosine. This technology may also be used tolabel proteins by binding to nitrogen and sulphur containing side chainsof amino acids. See, e.g., U.S. Pat. Nos. 5,580,990; 5,714,327; and5,985,566; and European Patent No. 0539466.

The binding of a probe to the marker sequence flanking the tandem repeatregion may be determined by hybridization as is well known in the art.Hybridization may be detected in real time or in non-real time.

TaqMan® probes (Heid, et al., 1996) use the fluorogenic 5′ exonucleaseactivity of Taq polymerase to measure the amount of target sequences incDNA samples. TaqMan® probes are oligonucleotides that contain a donorfluorophore usually at or near the 5′ base, and a quenching moietytypically at or near the 3′ base. The quencher moiety may be a dye suchas TAMRA or may be a non-fluorescent molecule such as4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). See Tyagi, et al.,16 Nature Biotechnology 49-53 (1998). When irradiated, the excitedfluorescent donor transfers energy to the nearby quenching moiety byFRET rather than fluorescing. Thus, the close proximity of the donor andquencher prevents emission of donor fluorescence while the probe isintact.

TaqMan® probes are designed to anneal to an internal region of a PCRproduct. When the polymerase (e.g., reverse transcriptase) replicates atemplate on which a TaqMan® probe is bound, its 5′ exonuclease activitycleaves the probe. This ends the activity of quencher (no FRET) and thedonor fluorophore starts to emit fluorescence which increases in eachcycle proportional to the rate of probe cleavage. Accumulation of PCRproduct is detected by monitoring the increase in fluorescence of thereporter dye (note that primers are not labeled). If the quencher is anacceptor fluorophore, then accumulation of PCR product can be detectedby monitoring the decrease in fluorescence of the acceptor fluorophore.

TaqMan® assay uses universal thermal cycling parameters and PCR reactionconditions. Because the cleavage occurs only if the probe hybridizes tothe target, the fluorescence detected originates from specificamplification. The process of hybridization and cleavage does notinterfere with the exponential accumulation of the product. One specificrequirement for fluorogenic probes is that there be no G at the 5′ end.A ‘G’ adjacent to the reporter dye quenches reporter fluorescence evenafter cleavage.

Other methods of probe hybridization detected in real time can be usedfor detecting amplification of mutated nucleic acids. For example, thecommercially available MGB Eclipse™ probes (Epoch Biosciences), which donot rely on a probe degradation can be used. MGB Eclipse™ probes work bya hybridization-triggered fluorescence mechanism. MGB Eclipse™ probeshave the Eclipse™ Dark Quencher and the MGB positioned at the 5′-end ofthe probe. The fluorophore is located on the 3′-end of the probe. Whenthe probe is in solution and not hybridized, the three dimensionalconformation brings the quencher into close proximity of thefluorophore, and the fluorescence is quenched. However, when the probeanneals to a target sequence, the probe is unfolded, the quencher ismoved from the fluorophore, and the resultant fluorescence can bedetected.

Suitable donor fluorophores include 6-carboxyfluorescein (FAM),tetrachloro-6-carboxyfluorescein (TET),2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and thelike. Suitable quenchers include tetra-methylcarboxyrhodamine (TAMRA)4-(4-dimethylaminophenylazo) benzoic acid (“DABCYL” or a DABCYL analog)and the like. Tetramethylrhodamine (TMR) or 5-carboxyrhodamine 6G (RHD)may be combined as donor fluorophores with DABCYL as quencher. MultiplexTaqMan® assays can be performed using multiple detectable labels eachcomprising a different donor and quencher combination. Probes fordetecting amplified sequence in real time may be stored frozen (−10° to−30° C.) as 100 M stocks. TaqMan® probes are available from AppliedBioSystems (4316032).

In a preferred embodiment, real time PCR is performed using TaqMan®probes in combination with a suitable amplification/analyzer such as theABI Prism 7900HT Sequence Detection System. The ABI PRISM® 7900HTSequence Detection System is a high-throughput real-time PCR system thatdetects and quantitates nucleic acid sequences. Briefly, TaqMan® probesspecific for each allele are included in the PCR assay. These probescontain a reporter dye at the 5′ end and a quencher dye at the 3′ end.Each allele specific probe is conjugated with a different fluorescentreporter dye. During PCR, the fluorescently labeled probes bindspecifically to their respective target sequences; the 5′ nucleaseactivity of Taq polymerase cleaves the reporter dye from the probe and afluorescent signal is generated. The increase in fluorescence signal isdetected only if the target sequence is complementary to the probe andis amplified during PCR. A mismatch between probe and target greatlyreduces the efficiency of probe hybridization and cleavage. The ABIPrism 7700HT or 7900HT Sequence detection System measures the increasein fluorescence during PCR thermal cycling, providing “real time”detection of PCR product accumulation.

Real Time detection on the ABI Prism 7900HT or 7900HT Sequence Detectormonitors fluorescence and calculates the measure of reporter signal, orRn value, during each PCR cycle. The threshold cycle, or Ct value, isthe cycle at which fluorescence intersects the threshold value. Thethreshold value is determined by the sequence detection system softwareor manually.

To minimize the potential for cross contamination, reagent and mastermixpreparation, specimen processing and PCR setup, and amplification anddetection are all carried out in physically separated areas. Inaddition, Uracil-N-Glycosylase is utilized (along with the incorporationof Uracil into PCR amplicons) to eliminate carry over contamination.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1 E746_A750 Deletion

Background:

Somatic mutations in the tyrosine kinase (TK) domain of the EGFR geneare associated with clinical response to TK inhibitors in patients withnon-small cell lung cancer (NSCLC). An assay that detects such mutationsin plasma provides a noninvasive procedure to assess suitability for TKinhibitor therapy. Described below are 1) the development of asensitivity assay to detect the E746_A750 deletion (E746_A750del) in theTK domain of EGFR in plasma; and 2) optimization of the assay for plasmaDNA extraction.

Methods:

The assay uses MseI to specifically digest wild-type (WT) genomic DNA(gDNA) to reduce non-specific amplification. After digestion, samplesare PCR-amplified using one unlabeled primer and one FAM-labeled primerspanning the E746_A750 deletion region. The fluorescence signal isdetected with an automated genetic analyzer. Serial dilution studieswere conducted using H1650 gDNA, which is a E746_A750 deletion cellline) diluted in WT gDNA after MseI digestion. To assess detection ofthe deletion in plasma, 3-4 mL of whole blood was spiked with 1-37 ngH1650 gDNA; gDNA from the separated plasma was then extracted by silicacolumn/2-propanol precipitation, digested with MseI, and amplified asabove. Several extraction methods (silica column, magnetic bead,phenol:chloroform, and 2-propanol precipitation) were evaluated usingpooled plasma samples and phosphate buffer solution (PBS) spiked with10-350 ng of gDNA.

Results:

Using a combined approach of digesting the WT EGFR allele followed bydeletion-specific fluorescent PCR, the equivalent of circa 1 copy of theE746_A750 deletion (10 pg gDNA) diluted to 0.001% could be detected.Furthermore, the E746_A750 deletion was successfully detected in ⅕ thefinal DNA volume (5 μl) in all spiked blood samples. In the DNAextraction method evaluation, the magnetic bead-based method yielded thehighest percent recovery of gDNA from PBS (69% recovery of the 10 ngsample). Phenol:chloroform extraction gave the highest yield with pooledplasma samples.

Conclusions:

The combination of an optimized DNA extraction method, clearing theplasma DNA sample of amplifiable WT DNA by restriction digestion, andmutation-specific fluorescent PCR provides a highly sensitive assay fordetection of somatic mutations in plasma.

Example 2 Preparation of and Sensitivity Studies for Detection of EGFRMutations

Serial dilution studies were conducted using H1650 gDNA (E746_A750deletion mutation cell line) diluted in wild-type gDNA with no treatmentof after MseI digestion. For each dilution, 10 pg of H1650 gDNA. H1650is a heterozygous cell line, thus, 50% of the DNA contributes to thedeletion allele and 50% to the wild-type allele) was spiked intowild-type gDNA at varying concentrations to yield 0.001%-10% of the exon19 deletion in the background of wild-type allele. Nucleic acid isspiked into a sample in order to control the amount of target nucleicacid in a sample and test the sensitivity of the assay to detect variousamounts, in particular low amounts, of target nucleic acid. The resultsshow that peaks were detected corresponding to the deletion mutant atlevels of 0.01% in non-treated samples and at least 0.001% when digestedwith MseI. These results are presented in Table 1.

TABLE 1 Exon 19 deletion tumor DNA spiked into purified DNA wild-typeDNA % Exon 19 DNA Amount per Peak Detected deletion reactionConcentration No MseI allele Wild-Type Exon 19 Deletion TreatmentTreated 10.00%  45 pg 10 pg (50% E746_A750del) Yes Yes 1.000% 495 pg 10pg (50% E746_A750del) Yes No 0.100%  5 ng 10 pg (50% E746_A750del) YesYes 0.010%  50 ng 10 pg (50% E746_A750del) Yes Yes 0.001% 500 ng 10 pg(50% E746_A750del) No Yes 0.000% 500 ng 0 pg No No *10 pg of tumor DNAwas spiked into wild-type DNA at varying concentrations, but since 1allele is wild-type, only half of the tumor DNA contributes to exon 19deletion DNA and the other half to the wild-type allele

To assess detection of the deletion in plasma, 3-4 mL whole blood wasspiked with 1-37 ng H1650 gDNA; gDNA from the separated plasma was thenextracted (QiaAmp DNA Blood Midi Kit), further concentrated by ethanolor isopropanol precipitation, and digested with MseI. The digested DNAwas then subjected to fluorescent PCR using deletion-specific primers.

TABLE 2 Exon 19 deletion tumor DNA spiked into blood Peak Detected Amtof Spiked No Precipitation Method H1650 gDNA Treatment Mse I TreatedEthanol Precipitation 37 ng No Yes  1 ng No No Isopropanol Precipitation37 ng NT Yes  1 ng NT Yes *All samples were first extracted using theQiaAmp DNA Blood Midi kit, then further precipitated by ethanol orisopropanol precipitation NT, Not tested

Example 3 Method Comparison of EGFR Mutation Detection

A method comparison was performed to demonstrate the sensitivity of theEGFR mutation detection assay disclosed herein (also referred to as theSanders method) which is designed to be able to detect mutations inplasma from NSCLC patients. In this study, the method disclosed hereinwas compared with two other methods (Asano, 2006 and Ohnishi, 2006) thatclaim high sensitivity for detecting E746_A750del. The Asano and Ohnishimethods were performed as described in the respective publications butincluded a fluorescent label on the forward primer. Amplificationproducts were then analyzed by capillary electrophoresis for fluorescentdetection of the PCR fragments using an ABI 3100 Genetic Analyzer. Table3 shows the expected and observed fragment sizes for each fragment.

TABLE 3 Expected and observed fragment sizes Method Fragment ExpectedSize Observed Size Sanders Wild-Type EGFR 197 194 E746_A750del 153 151Asano Wild-Type EGFR 138 135 E746_A750del 123 120 Ohnishi E746_A750del133 138 *Due to the mobility shift of PCR primer/products fluorescentlylabeled with FAM, some of the amplicons are slightly shifted from theexpected size in the ABI 3100 Genetic Analyzer.

The assay disclosed herein has been previously detected as little as 10pg of H1650 cell line DNA (circa 1 copy of the E746_A750del mutation) atas low as 0.001% in the background of the wild-type EGFR gene.Therefore, all three methods (Sanders, Asano and Ohnishi) were testedfor their ability to detect 10 pg of H1650 cell line DNA at levelsranging from 0.0005% to 10% in the background of the wild-type EGFR gene(Table 4).

TABLE 4 Sensitivity of Sanders, Asano, and Ohnishi methods for detectingthe E746_A750del mutation. Sanders Method Asano Method Ohnishi MethodWild-Type Deletion Peak Wild-Type Deletion Peak Deletion Peak PeakIntensity Intensity Peak Intensity Intensity % H1650 DNA Intensity (1/5)(120 bp)* (135 bp)* (138 bp)*    10% 937 0 5811 0 0    1% 989 166 7209195 0  0.10% 1395 152 2685 1746 0  0.01% 520 1395 1004 3682 0  0.001%820 796 97 7087 0 0.0005% 1184 2321 0 7338 167 0.0003% 699 3942 NT NT NT0.0002% 803 3324 NT NT NT 0.0001% 0 3798 0 4603 NT 0% (5 μg)  0 2287 03836 NT 0% (50 ng) 0 0 0 5280 0 *Sizes indicated are the actual size NT= Not Tested

The results of this comparison indicate that the Sanders method detectscirca 1 copy of the E746_A750del mutation in as little as 0.0005% in thebackground of the wild-type EGFR gene. (6.6 picograms (pg) is equivalentto one copy, so 10 pg is 1.5 copies, thus, circa is used to indicate theapproximate copy number.) The Asano method demonstrated strong peaks at0.01-10% levels and a weak peak at 0.001%, but no detectableamplification at 0.0005%. The Ohnishi method was unable to detect circa1 copy of the E746_A750del mutation as determined by the absence of anypeak from 0.001%-10% levels. However, a weak peak was observed in the0.0005% sample, although this is most likely attributed to backgroundamplification of the wild-type EGFR gene due to the very high levels ofwild-type DNA present in the sample.

The Asano and Ohnishi methods were also tested for their ability todetect the E746_A750del mutation at high and low copy numbers withoutinterfering wild-type DNA spike into the sample. Table 5 shows that theAsano method was successful at detecting both a high copy numbers (850pg=130 copies) and low copy numbers (10 pg=˜1 copy) of the mutation, asalso demonstrated above. However, while the Ohnishi method successfullydetected high copy numbers of the mutation, it failed at detecting lowcopy numbers.

TABLE 5 Detection of low and high copy numbers of the E746_A750delmutation. Asano Method Ohnishi Method Deletion Peak Wild Type DeletionPeak H1650 DNA Intensity Peak Intensity Intensity (pg/rxn) (120 bp)*(135 bp)* (138 bp)* 850 7345 0 2008 10 2422 0 0 *Sizes indicated are theactual size

The results presented in this study confirm that the Sanders assay fordetection of the exon 19 EGFR deletion (E746_A750del) utilizingrestriction digestion followed by deletion specific fluorescent PCR,demonstrates superior sensitivity over methods in the prior art, inparticular, the methods described by Asano and Ohnishi.

Example 4

Detection of E746_A750del Mutation in Plasma of a NSCLC Patient

The assay tested two separate DNA extractions from plasma of a singleNSCLC patient. Each extraction was digested with MseI and subsequentlysplit into 11 separate PCR reactions. Two reactions yielded positiveresults as shown in Table 6 below.

TABLE 6 E746_A750del Mutation in the Plasma of a NSCLC Patient PeakIntensity 500 μl Plasma 350 μl Plasma Primer set Aliquot # 1:10Undiluted 1:10 Undiluted E746_A750del 1 0 0 53 163 2 0 0 0 0 3 3568 79520 0 4 0 0 0 0 5 0 0 0 0 6 0 0 0 0 7 0 0 0 0 8 0 0 0 0 9 3971 7359 0 0 100 0 0 0 11 0 0 2518 6469 WT 1 0 — 0 —

Example 5 DNA Extraction Comparison

Detection of rare mutations in plasma is a difficult feat and isdependant on both the ability to detect very low amounts of the mutationamong large amounts of normal DNA and the ability to successfullyrecover the mutation from the patient plasma sample. Using a highquality DNA extraction method in conjunction with the methods andcompositions provided herein, particularly combining non-targetfragmentation with mutation specific primers further increases theability to detect low copy target nucleic acid in patient samples,particularly plasma.

To determine an optimal method of DNA extraction, eight DNA extractionmethods were evaluated to find a method that is superior for obtaininghigh yield DNA as well as providing DNA that is amenable to thedetection methods provided herein. Six plasma samples for eachextraction method were spiked with various nucleic acids including everyplasma sample being spiked with the EGFR exon 19 deletion (H1650 cellline gDNA) for subsequent evaluation of detection. All six plasmasamples were extracted using the eight methods that included two lysismethods using magnetic bead based methods (Agencourt Genfind™ and AmbionMagMax™), two column based methods (Qiagen QIAmp DNA Blood Mini Kit andthe automated Corbett DNA Xtractor™), two isopropanol precipitationbased methods (Gentra Puregene™ and Roche Cobas®), and twophenol:chloroform based methods (standard method and with EppendorfPhase Lock Gels®). The resulting yield was determined by picogreenfluorescent assay (Invitrogen Quant-iT™ PicoGreen® dsDNA QuantitationAssay) and the mean percent recovery of the six plasma samples wascalculated for each method. Comparison of these eight DNA extractionmethods revealed that three methods were clearly more efficient at DNArecovery than the other five (FIG. 14). These methods included AgencourtGenfind™, Roche Cobas® and phenol:chloroform extraction using EppendorfPhase Lock Gels®.

Ambion MagMax™ is an isolation kit which can be used to isolate RNA orDNA from serum, plasma, or any other biofluid. Agencourt Genfind™ is aDNA isolation for blood, serum, or plasma samples. Both involve sampledisruption with a lysis reagent followed by binding of the nucleic acidto magnetic beads (proprietary chemistry). The beads are then washedwith a series of buffers to reduce and/or eliminate proteins and othercontaminants from the sample in order to purify the nucleic acid. Thenucleic acid is then eluted off the beads to yield the final DNA sample.

QIAamp DNA Blood Mini Kit and Corbett DNA Xtractor™ are both columnbased methods. The QIAamp procedure used in these studies was manualwhile the Corbett was an automated system. Both involve disruption ofthe sample with a lysis reagent followed by binding to a silica column.The column containing bound nucleic acid is washed with a series of washbuffers and the nucleic acid is then eluted in the last step withelution buffer to yield the final sample.

Gentra Puregene™ and Roche Cobas® methods are crude extractions and bothincorporate treatment of plasma with a lysis reagent to disrupt thesample followed by isopropanol precipitation of DNA.

Phenol:Chloroform extraction involves separation of organic and aqueousphases of the plasma. The aqueous phase containing nucleic acid isisolated and re-extracted once more with phenol:chloroform. The aqueousphase is again isolated and the DNA is purified from this phase byisopropanol precipitation.

Phenol:Chloroform extraction using Phase Lock Gels® employs thephenol:chloroform procedure as described above, but once separated theaqueous and organic phases are separated by a solid gel. This allowsincreased recovery of the aqueous phase without contamination from theorganic phase which can lead to inhibition of PCR.

Further evaluations were performed to identify the best method from thethree yielding the highest amounts of DNA. For this comparison, 18% ofthe final DNA sample from each extraction was subjected to our EGFRmutation detection assay as described in the Examples above. The meanpeak intensity of the PCR products obtained corresponding to theE746_A750del EGFR mutation were calculated as an indicator of DNAquality and recovery of the spiked mutation (FIG. 15). Peak intensityvalues indicated that of the three methods tested, the AgencourtGenfind™ method provided the most robust amplification of spikedmutation.

Once the superior method was identified, further evaluation of the sixnucleic acid carrier conditions was performed to determine if eithercondition facilitated recovery of the spiked mutation. These nucleicacid carrier conditions included use of RNA carrier or no carrier at lowand high (100 nanograms (ng) spiked normal DNA) plasma DNA levels andthe use of normal DNA as a carrier (395 ng and 778 ng). To determineoptimal nucleic acid carrier conditions, dilutions of each sample wereanalyzed using our detection method. The concentration of mutant DNA inthe extracted samples was first estimated based on the calculatedpercent recovery for each sample. The equivalent of 15 pg of H1650 DNAwas used as the starting point and was further diluted 1:2 and 1:4. Allthree dilutions were analyzed for the presence of detectable spiked EGFRmutation. The samples employing RNA carrier were the only ones thatdetected the mutation in the 1:4 dilution indicating that spiking RNAcarrier during the extraction facilitates recovery of the mutationpresent in the plasma sample (FIG. 16). FIG. 16 provides the resultsfrom evaluation of nucleic acid spiking conditions. The six nucleic acidcarrier conditions include 1) no carrier, 2) no carrier plus 100 ng ofnormal DNA, 3) 1 μg of RNA carrier, 4) 1 μg of RNA carrier plus 100 ngof normal DNA, 5) 395 ng of normal DNA as a carrier, and 6) 778 ng ofnormal DNA as a carrier. Plasma samples spiked with 100 ng of normal DNAwere included to represent patient plasma samples containing highamounts of DNA and were spiked immediately after thawing of the plasmasample. The two samples containing 395 ng and 778 ng of normal DNA werespiked following sample lysis during the extraction, the point at whichnucleic acid carrier is to be added. Columns represent the mean peakintensity of E746_A750del PCR product.

The experiments performed in this study identified an optimal method forobtaining high yield DNA from plasma samples that is amenable todetection of rare mutations. This method was further improved byidentifying nucleic acid spiking conditions that facilitate the recoveryof mutations from the sample. Thus to improve the ability to recoverrare mutations in the plasma and to successfully detect such mutations,it was determined that the Agencourt Genfind™ method with a modificationto include addition of RNA carrier to the sample lysate is the choicemethod.

While Agencourt Genfind™ was the optimal extraction method in theseexperiments, any of the extraction methods tested can be used and areprovide as exemplary extraction methods that can be used in conjunctionwith the methods and compositions provided herein. In different hands,results may vary, but all are acceptable methods.

Example 6 Detection Sensitivity of L858R in Wild-Type Background

DNA samples were initially digested with restriction enzymes (NewEngland Biolabs) to cleave TTAA (MseI) and TGGCCA (MscI) recognitionsites targeting wild type EGFR sequences (Mse I for singleplexE746_A750del; Msc I for singleplex L858R; Mse I and Msc I for multiplexreactions) for 2 hours at 37° C. followed by 20 minute inactivation at65° C. Following restriction digestion, samples were amplified withAccuPrime™ Taq DNA Polymerase (Invitrogen) using E746_A750del and/orL858R mutation-specific primers (E746_A750del forward: 5′-[6FAM] CCC GTCGCT ATC AAA ACA TC-3′ (SEQ ID NO:1); E746_A750del reverse: 5′-ATG TGGAGA TGA GCA GGG TCT-3′ (SEQ ID NO:2); L858R forward: [6FAM] TCA CAG ATTTTG GGC GG-3′ (SEQ ID NO:7); L858R reverse: CCT GGT GTC AGG AAA ATGCT-3′ (SEQ ID NO:8)). In each set, the forward primer was labeled with5′-6FAM. For added specificity, the L858R forward primer contained alocked nucleic acid (LNA) in the −1 position corresponding to themutated base. Thermocycling conditions were as follows: denatured at 95°C. for 5 min; amplified with 40 cycles of 94° C. for 40 seconds, 55° C.(E746_A750del singleplex PCR) or 61.7° C. (L858R singleplex PCR andmultiplex PCR) for 1 minute, 72° C. for 1 minute; final extension at 72°C. for 7 minutes. E746_A750del PCR product yielded an expected size of153 bp and L858R PCR product yielded an expected size of 113 bp.

TABLE 7 Detection of L858R Mutation in the Background of Wild type EGFR.Mean Peak Intensity¹ % L858R (110 bp)²  0.001% 463 0.0005% 412 0.0003% —0.0002% — 0% (2.5 ug/rxn) — ¹Peak intensities <200 RFU are not reported²Size represents observed L858R peak size on ABI 3100 Genetic Analyzer

Several other mutation specific primers for detecting the L858R mutationwere tested but were not effective in determining the presence of themutant nucleic acid. The primers are provided in the table below:

TABLE 8 L858R Mutation Specific Primers. Mutant Base as a Locked SEQNucleic Acid ID NO: Primer Sequence (Yes or No) 13 ATCACAGATTTTGGGC GYes 14 CAAGATCACAGATTTTGGGC G No 15 ATGTCAAGATCACAGATTTTGGGC G No 16AGATCACAGATTTTGGGC G No 17 ATCACAGATTTTGGGC G G No 18 TCACAGATTTTGGGC GGG No 19 ATTTTGGGC G GGCCAAAC No 20 AGATTTTGGGC G GGCCA No 21ATCACAGATTTTGGGC G G Yes * The bolded underlined base is the location ofthe L858R mutant base.

Example 7 EGFR Mutation Detection in Paired Tissue and Plasma Samples

Paired FFPE tissue and plasma were obtained from 11 NSCLC donors withinformed consent (10 from Indivumed, Hamburg, Germany; 1 from GoodSamaritan Hospital, Kearney, Nebr.). DNA from FFPE tissue was analyzedby PCR followed by direct sequencing to determine mutation status of thepaired tissue/plasma donors. One donor provided two plasma samples (856and 3107) 1 year apart for analysis. In addition, 6 normal plasmasamples and 5 plasma samples spiked with E746_A750del or L858R mutationswere analyzed by the methods described herein.

Results of direct sequencing of FFPE tissue and multiplex fluorescentRF-PCR of plasma samples are presented in Table 3 along with tumor sizeand overall plasma DNA concentration. Overall 3/11 (27%) NSCLC donorshad mutation positive FFPE tissue, while the remaining 8 were negativefor the two mutations. Of the paired NSCLC samples, 83.3% (10/12) ofplasma samples demonstrated identical mutation status to the matchedFFPE tissue specimens. The two E746_A750del positive plasma samples weredrawn from the same donor 1 year apart, thus 81.8% (9/11) unique donorshad identical mutation status between paired samples. Notably, althoughthe overall plasma DNA concentration had decreased by nearly half, thelatter plasma specimen had twice as many positive wells as the specimendrawn 1 year previous (data not shown), suggesting the circulatingmutation concentration had increased in that time.

For the mutation spiked plasma samples, 100% of samples spiked with 100,200, and 300 pg of E746_A750del positive DNA (H1650 cell line) and 100%of samples spiked with 100 and 300 pg of L858R positive DNA (H1975 cellline) tested positive for their respective mutation. Furthermore, 6normal plasma specimens tested negative for either mutation as expected.

TABLE 9 EGFR Mutation Detection in Paired Tissue and Plasma SamplesTumor EGFR Mutation Detected Sample Diameter Plasma DNA Tissue Plasma ID(cm) Conc. (ng/mL) PCR/Direct Seq RF-PCR NSCLC  856¹ 6.5² 15.6E746_A750del E746_A750del 3107¹  6.5² 8.1 E746_A750del E746_A750del 3788.5 144 — — 401 5 20.6 — — 455 3.4 212 — — 477 3.5 54.0 — — 497 2.5 26.5L858R — 516 5.7 11.1 — — 532 3.5 41.5 — — 563 1.9 265 L858R — 631 2.9128 — — 662 10.5 386 — — E746_A750del Spiked 200 pg N/A 23.3 N/AE746_A750del 300 pg N/A 9.9 N/A E746_A750del 100 pg N/A 7.6 N/AE746_A750del L858R spiked 300 pg N/A 8.0 N/A L858R 100 pg N/A 6.4 N/AL858R Normal 4207  N/A 4.3 N/A — 2011  N/A 5.8 N/A — 2258  N/A 8.7 N/A —3391  N/A 7.4 N/A — 975 N/A 4.4 N/A — 725 N/A 7.1 N/A — ¹Sample IDs 856and 3107 are from the same patient. Sample ID 3107 was drawn 1 yearafter Sample ID 856. ²Tumor size measured 4.7 years prior to initialdraw and 5.7 years prior to second draw.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

That which is claimed is:
 1. A kit comprising: an oligonucleotide primerpair comprising a forward primer of SEQ NO:1 and a reverse primer of SEQNO:2 and restriction enzyme MseI; an oligonucleotide primer paircomprising a forward primer of SEQ NO:3 and a reverse primer of SEQ NO:4and restriction enzyme Mn1I; an oligonucleotide primer pair comprising aforward primer of SEQ NO:5 and a reverse primer of SEQ NO:6 andrestriction enzyme Mn1I; an oligonucleotide primer pair comprising aforward primer of SEQ NO:7 and a reverse primer of SEQ NO:8 and at leastone restriction enzyme selected from MscI or EaeI, or a combinationthereof; an oligonucleotide primer pair comprising a forward primer ofSEQ NO:9 and a reverse primer of SEQ NO:10 and restriction enzyme FatI;an oligonucleotide primer pair comprising a forward primer of SEQ NO:11and a reverse primer of SEQ NO:12 and restriction enzyme HpyCH4VI; or acombination thereof; at least one oligonucleotide of the primer pair islabeled with a detectable moiety.
 2. The kit of claim 1, wherein thedetectable moiety is a fluorescent dye.
 3. The kit of claim 1, whereindifferent pairs of primers in the kit are labeled with differentdistinguishable detectable moieties.
 4. The kit of claim 1, wherein theat least one primer pair comprises a forward primer and a reverse primerlabeled with different detectable moieties.
 5. The kit of claim 1,wherein the kit comprises the oligonucleotide primer pair SEQ ID NOs: 1and 2 and the restriction enzyme MseI.
 6. The kit of claim 1, whereinthe kit comprises the oligonucleotide primer pair SEQ ID NOs: 3 and 4and the restriction enzyme Mn1I.
 7. The kit of claim 1, wherein the kitcomprises the oligonucleotide primer pair SEQ ID NOs: 5 and 6 and therestriction enzyme Mn1I.
 8. The kit of claim 1, wherein the kitcomprises the oligonucleotide primer pair SEQ ID NOs: 7 and 8 and therestriction enzyme MscI or EaeI, or a combination thereof.
 9. The kit ofclaim 1, wherein the kit comprises the oligonucleotide primer pair SEQID NOs: 9 and 10 and the restriction enzyme FatI.
 10. The kit of claim1, wherein the kit comprises the oligonucleotide primer pair SEQ ID NOs:11 and 12 and the restriction enzyme HpyCH4VI.